CN109929029A - A method of improving recombinant human blood coagulation factor VII I high efficient expression - Google Patents

Abstract

The present invention relates to gene engineering technology fields, specifically a kind of raising recombinant human blood coagulation factor VII I (Recombinant Human Coagulation Factor VIII, referred to as " rhFVIII ") high efficient expression method, a kind of more particularly to method for improving gene recombinant human blood coagulation factor VIII expression quantity in mammalian cells.The present invention makes recombinant human blood coagulation factor VII I high efficient expression by the building and screening of selection Yu the design, recombinant expression plasmid and engineering cell of recombinant human blood coagulation factor VII I gene sequence and using certain density blood serum medium culture positive cell.The method overcome existing cell strain building difficulty, produce the disadvantages of complicated, expression quantity is lower, also have the characteristics that while reducing and researching and developing cost quick, easy, stable, short-cut method is provided for the industrialization transfer of recombinant human blood coagulation factor VII I, the exploitation and Therapy study for biological products have important research and application value.

Description

A method of improving recombinant human blood coagulation factor VII I high efficient expression

Technical field

The present invention relates to gene engineering technology field, specifically a kind of raising recombinant human blood coagulation factor VII I The method of (Recombinant Human Coagulation Factor VIII, referred to as " rhFVIII ") high efficient expression, especially It is related to a kind of method for improving recombinant human blood coagulation factor VII I expression quantity in mammalian cells.

Background technique

Haemophilia A is a kind of common X sex-linked inheritance hemorrhage, 85% is accounted in all hemophilia, morbidity is former Because being found in generation nineteen fifty, in generation nineteen sixty, starts blood extraction and is treated, and the 1970's realized that patient was in self-injection Treatment, the 1980's have broken out patient and have infected hepatitis virus and inhibition of HIV event, generation nineteen ninety start using recombined human blood coagulation because Son treatment recommends daily maintenence treatment to severe haemophiliac since the 2000's.Haemophiliachemophiliac average attack rate is about For 5-10/10 ten thousand, therefore estimate that there are about 400,000 patients in the whole world, there are about 100,000 for China.2014, hemophilia alliance (World Federation of Hemophilia, WFH) the hemophilia number investigation to register on the books is shown, the whole world about 17.85 ten thousand People, and China only has 11108 people and registers on the books.The hemophilia number of every ten thousand population of China is only 0.08, and countries in Europe About 1, the U.S. 0.54, India 0.14, first and last, there is also very big differences for Chinese hemophilia diagnosing and treating coverage rate Away from.

FDA has had been approved by 15 for treating the recombinant human blood coagulation factor VII I of haemophilia A at present (Recombinant Human Coagulation Factor VIII) product.Wherein Advate (hundred because stopping), Kogenate (Bai Keqi), Xyntha (Ren Jie) come into China.The Factor IX sale in the whole world has reached 7,000,000,000 international units year at present, Wherein 60% is genetic engineering recombinant blood coagulation factor VIII.RhFVIII has biological effect similar with haematogenous F8, but Yield is not supplied the generation for the haematogenous virus infection such as being influenced, and avoid HBV, HCV, HIV by blood source, and rhFVIII is in State hemophilia A patients' in use, have preferable effectiveness and reliability.However, approval enters the Bai Keqi and hundred of China Because only expensive, and China can't produce recombinant human blood coagulation factor at present, in addition hemophilia A cannot still cure at present, Therefore it needs to carry out replacement therapy using rhFVIII throughout one's life.So that cost issues become China overwhelming majority haemophilia A patient The major limiting factors for the treatment of, therefore, independent research production rF8 have positive meaning for the satisfaction of domestic patient demand Justice.

The BDD-F8 albumen of eukaryotic cell in vitro expression shows preferable blood coagulation bioactivity, but disadvantage is to be easy drop Solution.In order to improve the expression quantity of rhFVIII and reduce the degradation of albumen, scientist's once cotransfection in mammalian cells RhFVIII and VWF gene stablizes the expression of rhFVIII, but this this method has some limitations.The study found that VWF with RhFVIII coexpression causes exogenous DNA to enter cell overload, and cell strain building is more complicated, while cell co-expresses pressure, Cause the expression quantity of rhFVIII relatively low, stable factors cell strain is subsequent is also difficult to meet industry by high expression vWF filtered out etc. Change and requires.In addition, serum free medium is all used in the culture of cell expression at present, this is because serum-free can avoid serum batch Between the variation of quality, avoid serum from avoiding serum component to experimental study the toxic effect and serum source contact scar of cell Influence and the influence to purifying products.

Summary of the invention

The present invention to solve the above-mentioned problems, improves the expression quantity of rhFVIII, passes through recombinant human blood coagulation factor VII I gene The selection of sequence constructs and screens and provide a kind of culture containing serum with design, recombinant expression plasmid and engineering cell Method realizes the industrialized production of rhFVIII, by being groped serum content, has obtained optimal serum and has been added Amount, the additional amount of serum do not have an impact to isolating and purifying, while keeping the expression of rhFVIII simpler, efficiently.

In order to achieve the above object, the present invention adopts the following technical scheme:

A method of recombinant human blood coagulation factor VII I high efficient expression is improved, is included the following steps:

(1) building of recombination, the recombination include a segment signal peptide gene and rhFVIII gene；

(2) above-mentioned recombination segment is inserted into plasmid, obtains recombinant plasmid；

(3) above-mentioned recombinant plasmid is subjected to PvuI linearisation, electricity is gone in mammalian cell, identified and active survey It is fixed, screen positive cell strain；

(4) by it is above-mentioned screen positive cell strain is seeded in cell culture medium, cultivate 4-5 days, into cell culture medium External source fetal calf serum is added,

It collects culture solution and carries out determination of activity；It is characterized in that, the additional amount of the fetal calf serum is 2-6%.

Further, the rhFVIII gene order in step (1) of the present invention is SEQ ID NO:1.

Preferably, the signal peptide in step (1) of the present invention is reddish black former protein signal peptide (Azu) before killing element, nucleotides sequence It is classified as SEQ ID NO:4, obtaining recombination is Azu-rhFVIII, and nucleotides sequence is classified as SEQ ID NO:6.

Further, the amino acid sequence of recombination Azu-rhFVIII is SEQ ID NO:7.

Preferably, the plasmid in step (2) of the present invention is pXC17.4, and obtained recombinant expression plasmid is pXC17.4- Azu-rhFVIII。

Preferably, the mammalian cell in step (3) of the present invention is Chinese hamster ovary cell CHOK1SV-KO, is obtained Positive cell strain is CHOK1SV-KO/pXC17.4-Azu-rhFVIII.

Preferably, the cell culture medium in step (4) of the present invention is CD CHO AGT culture medium.

Further, the additional amount of fetal calf serum is 3-5% in step (4) of the present invention.

Preferably, the additional amount of fetal calf serum is 3% in step (4) of the present invention.

Further, invention also provides the screening techniques of positive cell strain are as follows: electricity is turned cell inoculation to no blood In clear culture medium, after 35-37 DEG C is cultivated 1 day, supernatant is abandoned in centrifugation, and preheated culture medium (50 μ are added toward cell precipitation Mol/L MSX CD CHO AGT culture medium) it is resuspended, re-suspension liquid is transferred to conical flask and continues to cultivate, is cultivated to after 10 days, sampling By PCR and activity identification, positive cell strain CHOK1SV-KO/pXC17.4-Azu-rhFVIII is screened to obtain.

The invention has the following advantages:

The present invention by building Azu-rhFVIII recombination, be then introduced into plasmid pXC17.4, and by transfect into Mammalian cell CHOK1SV-KO obtains positive cell strain by screening, in the fetal calf serum culture medium containing 2-6% concentration The positive cell strain is cultivated, recombinant human blood coagulation factor VII I is made to obtain stability and high efficiency expression and secretion.

Detailed description of the invention

Fig. 1 is the schematic diagram of recombinant expression plasmid pXC17.4-Azu-rhFVIII.

Fig. 2 is that positive cell Genomic PCR identifies schematic diagram.

Fig. 3 is rhFVIII determination of activity schematic diagram.

Specific embodiment

Term " recombinant human blood coagulation factor VII I " in the present invention and " rhFVIII " unless otherwise instructed, in the present invention For same meaning.

Below with reference to embodiment, the present invention will be further described, but institute's protection scope of the present invention is without being limited thereto.

The building of 1 recombination Azu-rhFVIII of embodiment

1. the full genome of optimization SP and recombinant human blood coagulation factor VII I (SP-rhFVIII) synthesize

According to document (L.Thim, B.Vandahl, J.Karlsson.et al (2010)and characterization of a new recombinant factor VIII(N8).Haemopbilia.16,349-359) The original signal peptide SP (nucleotides sequence is classified as SEQ ID NO:2) of announcement and the amino acid sequence (total 1464aa) of rhFVIII, According to the optimization principles of Chinese hamster ovary cell (CHO), the nucleic acid sequence of SP and rhFVIII are optimized.Simultaneously to sequence In restriction enzyme site carry out considered critical, HindIII, EcoRI, SalI, NotI, PvuI enzyme cannot be contained in the sequence of optimization Enzyme site, while HindIII and Kozak sequence is added to 5 ends ' of sequence, to 3 end ' of sequence add double terminator codons and Then EcoRI, the SP (nucleotides sequence is classified as SEQ ID NO:3) after being optimized obtain coding recombination SP-rhFVIII (nucleotides sequence is classified as SEQ ID NO:5) finally gives gene chemical synthesis company and carries out full genome synthesis (pUC57-SP- rhFVIII)。

2. the reddish black connection for killing former albumen (Azu) and rhFVIII sequence before element

Using the plasmid pUC57-SP-rhFVIII of synthesis as template, by upstream primer F1, (nucleotides sequence is classified as SEQ ID NO:8), containing XbaI restriction enzyme site；Downstream primer F2 (nucleotides sequence is classified as SEQ ID NO:9), contains BglIII digestion position Point.Other reaction solutions are added and carry out PCR, amplification obtains the N-terminal segment A (SEQ ID NO:11,896bp) containing rhFVIII.It connects Contained using HindIII and XbaI enzyme cutting and reddish black kill former albumen (Azurocidin preproprotein: abbreviation Azu) before element Plasmid pXC17.4-Azu-GJC, obtain contain part Azu segment B (SEQ ID NO:12,69bp).Next upstream is utilized Primers F 3 (nucleotides sequence is classified as SEQ ID NO:10), restriction enzyme site containing HindIII；Downstream primer F2 contains BglIII digestion Site carries out over-lap PCR by template of segment A and B, and amplification obtains N-terminal segment C (the SEQ ID containing Azu-rhFVIII NO:13,943bp), segment C is connected to pMD-18T by TA clone, interstitial granules pMD-18T-Azu-rhFVIII (N in acquisition End).

Middle interstitial granules pMD-18T-Azu-rhFVIII (N-terminal) obtains Azu-rhFVIII (N through HindIII and BglII digestion End) segment D (SEQ ID NO:14,937bp), the segment D of acquisition and pUC57-SP-rhFVIII plasmid through HindIII and The carrier segments E (SEQ ID NO:15,6117bp) of BglII double digestion recycling is attached, and obtains positive plasmid pUC57- Azu-rhFVIII(7054bp)。

The building of 2 recombinant expression plasmid pXC17.4-Azu-rhFVIII of embodiment

Obtained positive plasmid pUC57-Azu-rhFVIII is constructed by the way that after HindIII and EcoRII double digestion, recycling contains There is the segment F (SEQ ID NO:16,4413bp) of purpose Gene A zu-rhFVIII, with pXC17.4 through HindIII and EcoRII The carrier segments G (SEQ ID NO:17,6893bp) recycled after double digestion is attached, and screening obtains final recombinant expression plasmid PXC17.4-Azu-rhFVIII (11306 bp), the result is shown in Figure 1.Utilize final purpose Gene A zu-rhFVIII segment The both ends design primer of (4392bp) is sequenced, and sequencing result shows that clone gene segment is consistent with theory.

The acquisition of 3 positive cell CHOK1SV-KO/pXC17.4-Azu-rhFVIII of embodiment

1.pXC17.4-Azu-rhFVIII electrotransformation

Recombinant expression matter pXC17.4-Azu-rhFVIII is linearized using PvuI restriction enzyme site, and by purifying, Filtration sterilization and Sterility testing obtain linearization plasmid pXC17.4-Azu-rhFVIII (concentration 0.4ug/uL).It utilizes simultaneously CD CHO AGT culture medium (L-Gln containing 6mM) recovery and secondary culture CHOK1SV-KO cell, using Lonza company GS Xceed^TMMethod in Gene Expressions ystem specification prepares CHOK1SV-KO recipient cell, according to default journey Sequence (pulse 300V, 900 μ F, resistance ∞, exponential wave, 4mm gap) shocks by electricity linearization plasmid pXC17.4-Azu-rhFVIII It is transferred to CHOK1SV-KO recipient cell.

2.MSX pressurization screening

After electric shock, by the conical flask for the CD CHO AGT culture medium (without L-Gln) that liquid is transferred in the cup that shocks by electricity It is interior, it after mixing gently, is placed in shaking table, 35-37 DEG C, 140r/min, 8.0%CO₂Culture.After cultivating 1d, by all culture solutions It is transferred in 50mL centrifuge tube, supernatant is abandoned in centrifugation (200g, 5min), with preheated culture medium (50 μm of ol/L MSX CD CHO AGT culture medium) it is resuspended, it all moves in conical flask, continues to cultivate.About 10d or so is cultivated, sampling survey vigor is close daily later Degree, until vigor is higher (such as larger than 90%), then preparation freezes and continues secondary culture.

3. positive cell screening and identification

After MSX pressurization screening, the recipient cell of untransfected is being lacked due to lacking endogenic glutamine synthelase Can not be grown in the culture medium of weary L-Gln, the successful engineering cell of plasmid transfection will continued growth go down.Simultaneously by drawing Object F3, restriction enzyme site containing HindIII；Primers F 2 is carried out containing BglIII restriction enzyme site by template of the cellular genome of extraction PCR amplification obtains the DNA fragmentation C being consistent with theoretical size (943bp), as a result sees Fig. 2.Pass through sequencing display amplification piece simultaneously Section is consistent with theory, illustrates that pXC17.4-Azu-rhFVIII is integrated on CHOK1SV-KO genome.Additionally by analysis detection Cells and supernatant, CHOK1SV-KO recipient cell supernatant is without rhFVIII activity, engineering cell CHOK1SV-KO/ as the result is shown PXC17.4-Azu-rhFVIII has rhFVIII active, is shown in Table 1 in detail.

The high efficient expression of 4 recombinant human blood coagulation factor VII I of embodiment

Recovered with the positive cell for screening and identifying, culture 3-4 days after, until cell density reach 1-2 × 10⁶Cells/mL is passed on, and culture to cell density reaches 1~2 × 10⁶Cells/mL obtains seed liquor.By the kind of acquisition Sub- liquid is inoculated into the 1L shaking flask containing CD CHO AGT culture medium, cultivates 4~5 days, the cell density of cell liquid is made to reach 1 ~5 × 10⁶cells/mL；Then external source fetal calf serum is added into cell liquid.Fetal calf serum additional amount and corresponding recombined human Blood coagulation factor VIII expression activity data are shown in Table 1, by the expression quantity of rhFVIII in experimental data, select fetal calf serum addition Optimal addn be 2-6%.

The purifying of 5 recombinant human blood coagulation factor VII I of embodiment and Activity determination

1. the purifying of recombinant human blood coagulation factor VII I

After cell culture fluid passes through low-speed centrifugal (200g, 5min), the supernatant of acquisition further passes through high speed refrigerated centrifuge (8000rpm, 5min, 4 DEG C) obtains purer supernatant.

The determination of activity of 2.rhFVIII

In Ca₂ ⁺Under the conditions of existing for phosphatide, Fator X generates Fator Xa, this activation by Fator IXa activation Reaction can greatly be amplified by rhFVIII, by optimizing Ca₂ ⁺, phosphatide, Fator IXa and excessive Fator X, The activation rate of Fator X is linear related to rhFVIII.

RhFVIII determination of activity schematic diagram is shown in Fig. 3.

Chromophoric substrate S-2765 (N-a-Z-D-Arg-Gly-Arg-pNA) is the specificity for activating the X factor (factor Xa) Substrate.S-2765 can release chromophore paranitroanilinum (pNA) by Xa factor catalyzing hydrolysis.PNA can be in 405nm wavelength Place detects that the absorption value detected is linear related to rhFVIII, and then can calculate the activity of rhFVIII.

1 recombinant human blood coagulation factor VII I determination of activity result of table

SEQUENCE LISTING

<110>Guangdong Dongyang Guang Pharmaceutical Co., Ltd

<120>a kind of method for improving recombinant human blood coagulation factor VII I high efficient expression

<130> 2017

<160> 17

<170> PatentIn version 3.5

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<211> 4335

<212> DNA

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gccacaaggc ggtactatct gggcgctgtg gaactgtcat gggattacat gcagtccgac 60

ctgggagagc tgccagtgga cgctcgattt ccccctcgtg tccctaagag cttcccattc 120

aacacatctg tggtctataa gaaaacactg ttcgtggagt ttactgatca cctgttcaac 180

atcgccaagc ctaggccacc ctggatggga ctgctgggac caactatcca ggctgaggtg 240

tacgacaccg tggtcattac actgaaaaac atggcatcac acccagtgtc cctgcatgcc 300

gtgggggtct cttactggaa ggctagtgaa ggtgcagagt atgacgatca gacatctcag 360

cgggaaaaag aggacgataa ggtgtttccc ggcggatccc atacttacgt gtggcaggtc 420

ctgaaagaga atggccccat ggcttctgat cctctgtgcc tgacctactc ttatctgagt 480

cacgtggacc tggtcaagga tctgaactcc gggctgatcg gtgcactgct ggtgtgcaga 540

gaagggagcc tggccaagga gaaaacccag acactgcata agttcattct gctgttcgcc 600

gtgtttgacg agggtaaatc atggcactcc gagaccaaga actccctgat gcaggacaga 660

gatgcagcta gcgcccgagc ttggccaaaa atgcatacag tgaacggcta cgtcaatagg 720

agtctgcccg gcctgatcgg atgtcaccgg aagtccgtgt attggcatgt catcgggatg 780

ggtaccacac ccgaagtgca ctctattttc ctggagggac atacctttct ggtccgaaac 840

caccgtcagg caagcctgga gatctctcct attaccttcc tgacagccca gactctgctg 900

atggatctgg ggcagttcct gctgttttgc cacatctcca gccaccagca tgatggtatg 960

gaggcatacg tgaaagtgga ctcctgtccc gaggaacctc agctgagaat gaagaacaat 1020

gaggaagccg aagactatga cgatgacctg actgactccg agatggatgt ggtccgcttc 1080

gatgacgata actccccctc cttcatccag attcgaagcg tggccaagaa acaccccaag 1140

acctgggtcc attacatcgc agccgaggaa gaggactggg attatgcacc actggtgctg 1200

gcaccagacg acaggtccta caaatctcag tatctgaaca atgggcctca gaggatcggt 1260

cggaagtaca agaaagtgcg attcatggcc tataccgatg aaacatttaa gactcgtgaa 1320

gctatccagc acgagtccgg gattctgggt ccactgctgt acggcgaagt gggagacact 1380

ctgctgatca tcttcaagaa ccaggctagc aggccttaca atatctatcc acatggcatt 1440

accgatgtgc ggcctctgta ctctagacgc ctgccaaagg gcgtcaaaca cctgaaggac 1500

ttcccaatcc tgcccggaga aatcttcaag tataaatgga ctgtcaccgt cgaggatgga 1560

cccactaagt ctgaccctag gtgcctgacc cggtactatt ctagtttcgt gaatatggaa 1620

agggatctgg cctccgggct gatcggtcca ctgctgattt gttacaaaga gtctgtggat 1680

cagcgaggca accagatcat gagtgacaag cgtaatgtga ttctgttctc cgtctttgac 1740

gaaaacagaa gctggtatct gaccgagaac atccagaggt tcctgcctaa tccagcaggc 1800

gtgcagctgg aagatcccga gtttcaggcc tcaaacatca tgcattccat taatggatac 1860

gtgttcgaca gtctgcagct gtcagtgtgc ctgcacgagg tcgcctactg gtatatcctg 1920

tccattgggg ctcagacaga tttcctgtcc gtgttcttta gcggttacac tttcaagcat 1980

aagatggtct acgaggacac actgactctg ttcccttttt ctggcgaaac cgtgtttatg 2040

agtatggaga atccaggcct gtggatcctg ggatgccaca acagcgattt ccgaaatcgt 2100

ggcatgactg ctctgctgaa agtgtcatcc tgtgacaaga acaccggaga ctactatgaa 2160

gatagctacg aggacatctc tgcttatctg ctgagtaaga acaatgcaat tgaacccagg 2220

tcttttagtc agaactctcg gcatcctagt cagaatcctc cagtgctgaa gagacaccag 2280

cgcgagatca ctagaactac cctgcagagt gatcaggaag agatcgacta cgacgatacc 2340

atttccgtgg aaatgaagaa agaggacttc gatatctatg acgaagatga gaaccagagt 2400

cctagatcat tccagaagaa aacccgccat tactttattg ctgcagtgga gcgcctgtgg 2460

gattatggga tgagctctag tccccacgtg ctgaggaatc gggcccagtc aggttccgtc 2520

cctcagttca agaaagtggt cttccaggag tttacagacg gcagctttac tcagcctctg 2580

tacagaggag aactgaacga gcacctgggc ctgctgggac catatatccg cgccgaagtg 2640

gaggataaca ttatggtcac cttccgaaat caggcttcac gtccctactc cttttattca 2700

tccctgatct cttacgaaga ggaccagcga cagggcgctg aaccccgtaa aaacttcgtg 2760

aagcctaatg agaccaaaac atacttttgg aaggtgcagc accatatggc ccctacaaaa 2820

gacgagttcg attgcaaggc atgggcctat ttttcagacg tggatctgga gaaggacgtg 2880

cactctgggc tgatcggtcc tctgctggtg tgccatacta acaccctgaa tccagctcac 2940

ggcaggcagg tgacagtcca ggagttcgca ctgttcttta ccatctttga tgagacaaag 3000

tcctggtact tcactgaaaa catggagcga aattgccgtg ctccatgtaa tattcagatg 3060

gaagacccca ccttcaagga gaactaccgg tttcatgcaa tcaatggcta tattatggat 3120

acactgccag gactggtgat ggcccaggac cagagaatcc gctggtacct gctgtccatg 3180

ggcagcaacg agaatatcca cagcattcat ttctctggac acgtgtttac agtccgcaag 3240

aaagaagagt ataaaatggc cctgtacaac ctgtatccag gggtgttcga aactgtcgag 3300

atgctgccca gcaaggctgg tatctggcga gtggaatgcc tgattgggga gcacctgcat 3360

gcaggcatgt caaccctgtt tctggtgtac tccaataagt gtcagacacc actggggatg 3420

gccagcggtc atatccgaga tttccagatt accgcttctg gccagtacgg acagtgggcc 3480

cccaagctgg ctaggctgca ctatagcggc tctatcaacg cctggagtac caaagagccc 3540

ttttcatgga ttaaggtgga cctgctggct cctatgatca ttcatgggat caaaacacag 3600

ggtgcacggc agaagttcag ctctctgtac atcagccagt tcatcatcat gtactctctg 3660

gatggaaaga aatggcagac atacagaggc aatagcacag gaactctgat ggtgttcttt 3720

ggcaacgtgg acagttcagg aatcaagcac aacattttca atccccctat cattgctagg 3780

tacatccggc tgcaccctac ccattattct attagaagta cactgcgcat ggaactgatg 3840

gggtgcgatc tgaacagttg ttcaatgcca ctgggtatgg agagtaaggc aatctcagac 3900

gcccagatta ccgcttccag ctacttcact aatatgtttg ctacctggtc ccctagcaaa 3960

gcaagactgc atctgcaggg caggagcaac gcatggcggc cacaggtgaa caatcccaag 4020

gagtggctgc aggtcgattt tcagaaaact atgaaggtga ccggcgtcac aactcaggga 4080

gtgaaatccc tgctgactag catgtatgtc aaggagttcc tgatctctag ttcacaggac 4140

ggacaccagt ggaccctgtt ctttcagaac gggaaggtga aagtcttcca gggtaatcag 4200

gatagtttta cacccgtggt caactcactg gacccacccc tgctgactag atacctgcgc 4260

atccaccctc agtcttgggt gcatcagatt gctctgagga tggaagtcct gggctgtgag 4320

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<223>SP+rhFVIII after optimizing

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atgcagatcg agctgagcac ctgcttcttc ctgtgcctgc tgcggttctg cttttctgcc 60

acaaggcggt actatctggg cgctgtggaa ctgtcatggg attacatgca gtccgacctg 120

ggagagctgc cagtggacgc tcgatttccc cctcgtgtcc ctaagagctt cccattcaac 180

acatctgtgg tctataagaa aacactgttc gtggagttta ctgatcacct gttcaacatc 240

gccaagccta ggccaccctg gatgggactg ctgggaccaa ctatccaggc tgaggtgtac 300

gacaccgtgg tcattacact gaaaaacatg gcatcacacc cagtgtccct gcatgccgtg 360

ggggtctctt actggaaggc tagtgaaggt gcagagtatg acgatcagac atctcagcgg 420

gaaaaagagg acgataaggt gtttcccggc ggatcccata cttacgtgtg gcaggtcctg 480

aaagagaatg gccccatggc ttctgatcct ctgtgcctga cctactctta tctgagtcac 540

gtggacctgg tcaaggatct gaactccggg ctgatcggtg cactgctggt gtgcagagaa 600

gggagcctgg ccaaggagaa aacccagaca ctgcataagt tcattctgct gttcgccgtg 660

tttgacgagg gtaaatcatg gcactccgag accaagaact ccctgatgca ggacagagat 720

gcagctagcg cccgagcttg gccaaaaatg catacagtga acggctacgt caataggagt 780

ctgcccggcc tgatcggatg tcaccggaag tccgtgtatt ggcatgtcat cgggatgggt 840

accacacccg aagtgcactc tattttcctg gagggacata cctttctggt ccgaaaccac 900

cgtcaggcaa gcctggagat ctctcctatt accttcctga cagcccagac tctgctgatg 960

gatctggggc agttcctgct gttttgccac atctccagcc accagcatga tggtatggag 1020

gcatacgtga aagtggactc ctgtcccgag gaacctcagc tgagaatgaa gaacaatgag 1080

gaagccgaag actatgacga tgacctgact gactccgaga tggatgtggt ccgcttcgat 1140

gacgataact ccccctcctt catccagatt cgaagcgtgg ccaagaaaca ccccaagacc 1200

tgggtccatt acatcgcagc cgaggaagag gactgggatt atgcaccact ggtgctggca 1260

ccagacgaca ggtcctacaa atctcagtat ctgaacaatg ggcctcagag gatcggtcgg 1320

aagtacaaga aagtgcgatt catggcctat accgatgaaa catttaagac tcgtgaagct 1380

atccagcacg agtccgggat tctgggtcca ctgctgtacg gcgaagtggg agacactctg 1440

ctgatcatct tcaagaacca ggctagcagg ccttacaata tctatccaca tggcattacc 1500

gatgtgcggc ctctgtactc tagacgcctg ccaaagggcg tcaaacacct gaaggacttc 1560

ccaatcctgc ccggagaaat cttcaagtat aaatggactg tcaccgtcga ggatggaccc 1620

actaagtctg accctaggtg cctgacccgg tactattcta gtttcgtgaa tatggaaagg 1680

gatctggcct ccgggctgat cggtccactg ctgatttgtt acaaagagtc tgtggatcag 1740

cgaggcaacc agatcatgag tgacaagcgt aatgtgattc tgttctccgt ctttgacgaa 1800

aacagaagct ggtatctgac cgagaacatc cagaggttcc tgcctaatcc agcaggcgtg 1860

cagctggaag atcccgagtt tcaggcctca aacatcatgc attccattaa tggatacgtg 1920

ttcgacagtc tgcagctgtc agtgtgcctg cacgaggtcg cctactggta tatcctgtcc 1980

attggggctc agacagattt cctgtccgtg ttctttagcg gttacacttt caagcataag 2040

atggtctacg aggacacact gactctgttc cctttttctg gcgaaaccgt gtttatgagt 2100

atggagaatc caggcctgtg gatcctggga tgccacaaca gcgatttccg aaatcgtggc 2160

atgactgctc tgctgaaagt gtcatcctgt gacaagaaca ccggagacta ctatgaagat 2220

agctacgagg acatctctgc ttatctgctg agtaagaaca atgcaattga acccaggtct 2280

tttagtcaga actctcggca tcctagtcag aatcctccag tgctgaagag acaccagcgc 2340

gagatcacta gaactaccct gcagagtgat caggaagaga tcgactacga cgataccatt 2400

tccgtggaaa tgaagaaaga ggacttcgat atctatgacg aagatgagaa ccagagtcct 2460

agatcattcc agaagaaaac ccgccattac tttattgctg cagtggagcg cctgtgggat 2520

tatgggatga gctctagtcc ccacgtgctg aggaatcggg cccagtcagg ttccgtccct 2580

cagttcaaga aagtggtctt ccaggagttt acagacggca gctttactca gcctctgtac 2640

agaggagaac tgaacgagca cctgggcctg ctgggaccat atatccgcgc cgaagtggag 2700

gataacatta tggtcacctt ccgaaatcag gcttcacgtc cctactcctt ttattcatcc 2760

ctgatctctt acgaagagga ccagcgacag ggcgctgaac cccgtaaaaa cttcgtgaag 2820

cctaatgaga ccaaaacata cttttggaag gtgcagcacc atatggcccc tacaaaagac 2880

gagttcgatt gcaaggcatg ggcctatttt tcagacgtgg atctggagaa ggacgtgcac 2940

tctgggctga tcggtcctct gctggtgtgc catactaaca ccctgaatcc agctcacggc 3000

aggcaggtga cagtccagga gttcgcactg ttctttacca tctttgatga gacaaagtcc 3060

tggtacttca ctgaaaacat ggagcgaaat tgccgtgctc catgtaatat tcagatggaa 3120

gaccccacct tcaaggagaa ctaccggttt catgcaatca atggctatat tatggataca 3180

ctgccaggac tggtgatggc ccaggaccag agaatccgct ggtacctgct gtccatgggc 3240

agcaacgaga atatccacag cattcatttc tctggacacg tgtttacagt ccgcaagaaa 3300

gaagagtata aaatggccct gtacaacctg tatccagggg tgttcgaaac tgtcgagatg 3360

ctgcccagca aggctggtat ctggcgagtg gaatgcctga ttggggagca cctgcatgca 3420

ggcatgtcaa ccctgtttct ggtgtactcc aataagtgtc agacaccact ggggatggcc 3480

agcggtcata tccgagattt ccagattacc gcttctggcc agtacggaca gtgggccccc 3540

aagctggcta ggctgcacta tagcggctct atcaacgcct ggagtaccaa agagcccttt 3600

tcatggatta aggtggacct gctggctcct atgatcattc atgggatcaa aacacagggt 3660

gcacggcaga agttcagctc tctgtacatc agccagttca tcatcatgta ctctctggat 3720

ggaaagaaat ggcagacata cagaggcaat agcacaggaa ctctgatggt gttctttggc 3780

aacgtggaca gttcaggaat caagcacaac attttcaatc cccctatcat tgctaggtac 3840

atccggctgc accctaccca ttattctatt agaagtacac tgcgcatgga actgatgggg 3900

tgcgatctga acagttgttc aatgccactg ggtatggaga gtaaggcaat ctcagacgcc 3960

cagattaccg cttccagcta cttcactaat atgtttgcta cctggtcccc tagcaaagca 4020

agactgcatc tgcagggcag gagcaacgca tggcggccac aggtgaacaa tcccaaggag 4080

tggctgcagg tcgattttca gaaaactatg aaggtgaccg gcgtcacaac tcagggagtg 4140

aaatccctgc tgactagcat gtatgtcaag gagttcctga tctctagttc acaggacgga 4200

caccagtgga ccctgttctt tcagaacggg aaggtgaaag tcttccaggg taatcaggat 4260

agttttacac ccgtggtcaa ctcactggac ccacccctgc tgactagata cctgcgcatc 4320

caccctcagt cttgggtgca tcagattgct ctgaggatgg aagtcctggg ctgtgaggca 4380

caggacctgt at 4392

<210> 6

<211> 4392

<212> DNA

<213> artificial sequence

<220>

<223> Azu+rhFVIII

<400> 6

atgaccagac tgaccgtgct ggctctgctg gccggactgc tggcttcttc tagagccgcc 60

acaaggcggt actatctggg cgctgtggaa ctgtcatggg attacatgca gtccgacctg 120

ggagagctgc cagtggacgc tcgatttccc cctcgtgtcc ctaagagctt cccattcaac 180

acatctgtgg tctataagaa aacactgttc gtggagttta ctgatcacct gttcaacatc 240

gccaagccta ggccaccctg gatgggactg ctgggaccaa ctatccaggc tgaggtgtac 300

gacaccgtgg tcattacact gaaaaacatg gcatcacacc cagtgtccct gcatgccgtg 360

ggggtctctt actggaaggc tagtgaaggt gcagagtatg acgatcagac atctcagcgg 420

gaaaaagagg acgataaggt gtttcccggc ggatcccata cttacgtgtg gcaggtcctg 480

aaagagaatg gccccatggc ttctgatcct ctgtgcctga cctactctta tctgagtcac 540

gtggacctgg tcaaggatct gaactccggg ctgatcggtg cactgctggt gtgcagagaa 600

gggagcctgg ccaaggagaa aacccagaca ctgcataagt tcattctgct gttcgccgtg 660

tttgacgagg gtaaatcatg gcactccgag accaagaact ccctgatgca ggacagagat 720

gcagctagcg cccgagcttg gccaaaaatg catacagtga acggctacgt caataggagt 780

ctgcccggcc tgatcggatg tcaccggaag tccgtgtatt ggcatgtcat cgggatgggt 840

accacacccg aagtgcactc tattttcctg gagggacata cctttctggt ccgaaaccac 900

cgtcaggcaa gcctggagat ctctcctatt accttcctga cagcccagac tctgctgatg 960

gatctggggc agttcctgct gttttgccac atctccagcc accagcatga tggtatggag 1020

gcatacgtga aagtggactc ctgtcccgag gaacctcagc tgagaatgaa gaacaatgag 1080

gaagccgaag actatgacga tgacctgact gactccgaga tggatgtggt ccgcttcgat 1140

gacgataact ccccctcctt catccagatt cgaagcgtgg ccaagaaaca ccccaagacc 1200

tgggtccatt acatcgcagc cgaggaagag gactgggatt atgcaccact ggtgctggca 1260

ccagacgaca ggtcctacaa atctcagtat ctgaacaatg ggcctcagag gatcggtcgg 1320

aagtacaaga aagtgcgatt catggcctat accgatgaaa catttaagac tcgtgaagct 1380

atccagcacg agtccgggat tctgggtcca ctgctgtacg gcgaagtggg agacactctg 1440

ctgatcatct tcaagaacca ggctagcagg ccttacaata tctatccaca tggcattacc 1500

gatgtgcggc ctctgtactc tagacgcctg ccaaagggcg tcaaacacct gaaggacttc 1560

ccaatcctgc ccggagaaat cttcaagtat aaatggactg tcaccgtcga ggatggaccc 1620

actaagtctg accctaggtg cctgacccgg tactattcta gtttcgtgaa tatggaaagg 1680

gatctggcct ccgggctgat cggtccactg ctgatttgtt acaaagagtc tgtggatcag 1740

cgaggcaacc agatcatgag tgacaagcgt aatgtgattc tgttctccgt ctttgacgaa 1800

aacagaagct ggtatctgac cgagaacatc cagaggttcc tgcctaatcc agcaggcgtg 1860

cagctggaag atcccgagtt tcaggcctca aacatcatgc attccattaa tggatacgtg 1920

ttcgacagtc tgcagctgtc agtgtgcctg cacgaggtcg cctactggta tatcctgtcc 1980

attggggctc agacagattt cctgtccgtg ttctttagcg gttacacttt caagcataag 2040

atggtctacg aggacacact gactctgttc cctttttctg gcgaaaccgt gtttatgagt 2100

atggagaatc caggcctgtg gatcctggga tgccacaaca gcgatttccg aaatcgtggc 2160

atgactgctc tgctgaaagt gtcatcctgt gacaagaaca ccggagacta ctatgaagat 2220

agctacgagg acatctctgc ttatctgctg agtaagaaca atgcaattga acccaggtct 2280

tttagtcaga actctcggca tcctagtcag aatcctccag tgctgaagag acaccagcgc 2340

gagatcacta gaactaccct gcagagtgat caggaagaga tcgactacga cgataccatt 2400

tccgtggaaa tgaagaaaga ggacttcgat atctatgacg aagatgagaa ccagagtcct 2460

agatcattcc agaagaaaac ccgccattac tttattgctg cagtggagcg cctgtgggat 2520

tatgggatga gctctagtcc ccacgtgctg aggaatcggg cccagtcagg ttccgtccct 2580

cagttcaaga aagtggtctt ccaggagttt acagacggca gctttactca gcctctgtac 2640

agaggagaac tgaacgagca cctgggcctg ctgggaccat atatccgcgc cgaagtggag 2700

gataacatta tggtcacctt ccgaaatcag gcttcacgtc cctactcctt ttattcatcc 2760

ctgatctctt acgaagagga ccagcgacag ggcgctgaac cccgtaaaaa cttcgtgaag 2820

cctaatgaga ccaaaacata cttttggaag gtgcagcacc atatggcccc tacaaaagac 2880

gagttcgatt gcaaggcatg ggcctatttt tcagacgtgg atctggagaa ggacgtgcac 2940

tctgggctga tcggtcctct gctggtgtgc catactaaca ccctgaatcc agctcacggc 3000

aggcaggtga cagtccagga gttcgcactg ttctttacca tctttgatga gacaaagtcc 3060

tggtacttca ctgaaaacat ggagcgaaat tgccgtgctc catgtaatat tcagatggaa 3120

gaccccacct tcaaggagaa ctaccggttt catgcaatca atggctatat tatggataca 3180

ctgccaggac tggtgatggc ccaggaccag agaatccgct ggtacctgct gtccatgggc 3240

agcaacgaga atatccacag cattcatttc tctggacacg tgtttacagt ccgcaagaaa 3300

gaagagtata aaatggccct gtacaacctg tatccagggg tgttcgaaac tgtcgagatg 3360

ctgcccagca aggctggtat ctggcgagtg gaatgcctga ttggggagca cctgcatgca 3420

ggcatgtcaa ccctgtttct ggtgtactcc aataagtgtc agacaccact ggggatggcc 3480

agcggtcata tccgagattt ccagattacc gcttctggcc agtacggaca gtgggccccc 3540

aagctggcta ggctgcacta tagcggctct atcaacgcct ggagtaccaa agagcccttt 3600

tcatggatta aggtggacct gctggctcct atgatcattc atgggatcaa aacacagggt 3660

gcacggcaga agttcagctc tctgtacatc agccagttca tcatcatgta ctctctggat 3720

ggaaagaaat ggcagacata cagaggcaat agcacaggaa ctctgatggt gttctttggc 3780

aacgtggaca gttcaggaat caagcacaac attttcaatc cccctatcat tgctaggtac 3840

atccggctgc accctaccca ttattctatt agaagtacac tgcgcatgga actgatgggg 3900

tgcgatctga acagttgttc aatgccactg ggtatggaga gtaaggcaat ctcagacgcc 3960

cagattaccg cttccagcta cttcactaat atgtttgcta cctggtcccc tagcaaagca 4020

agactgcatc tgcagggcag gagcaacgca tggcggccac aggtgaacaa tcccaaggag 4080

tggctgcagg tcgattttca gaaaactatg aaggtgaccg gcgtcacaac tcagggagtg 4140

aaatccctgc tgactagcat gtatgtcaag gagttcctga tctctagttc acaggacgga 4200

caccagtgga ccctgttctt tcagaacggg aaggtgaaag tcttccaggg taatcaggat 4260

agttttacac ccgtggtcaa ctcactggac ccacccctgc tgactagata cctgcgcatc 4320

caccctcagt cttgggtgca tcagattgct ctgaggatgg aagtcctggg ctgtgaggca 4380

caggacctgt at 4392

<210> 7

<211> 1464

<212> PRT

<213> artificial sequence

<220>

<223>Azu+rhFVIII amino acid sequence

<400> 7

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro

755 760 765

Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg

770 775 780

Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile

785 790 795 800

Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu

805 810 815

Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile

820 825 830

Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His

835 840 845

Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys

850 855 860

Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr

865 870 875 880

Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg

885 890 895

Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser

900 905 910

Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln

915 920 925

Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr

930 935 940

Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp

945 950 955 960

Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu

965 970 975

Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr

980 985 990

Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe

995 1000 1005

Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe

1010 1015 1020

Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln

1025 1030 1035

Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala Ile

1040 1045 1050

Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln

1055 1060 1065

Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu

1070 1075 1080

Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg

1085 1090 1095

Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly

1100 1105 1110

Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp

1115 1120 1125

Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly Met Ser

1130 1135 1140

Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly

1145 1150 1155

Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly

1160 1165 1170

Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser

1175 1180 1185

Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile

1190 1195 1200

Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr

1205 1210 1215

Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe

1220 1225 1230

Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg

1235 1240 1245

Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp

1250 1255 1260

Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala

1265 1270 1275

Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr

1280 1285 1290

Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met

1295 1300 1305

Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr

1310 1315 1320

Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser

1325 1330 1335

Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro

1340 1345 1350

Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys

1355 1360 1365

Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu

1370 1375 1380

Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser Gln

1385 1390 1395

Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys

1400 1405 1410

Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser

1415 1420 1425

Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln

1430 1435 1440

Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys

1445 1450 1455

Glu Ala Gln Asp Leu Tyr

1460

<210> 8

<211> 50

<212> DNA

<213> artificial sequence

<220>

<223>primers F 1

<400> 8

ggccggactg ctggcttctt ctagagccgc cacaaggcgg tactatctgg 50

<210> 9

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223>primers F 2

<400> 9

ggaagatctc caggcttgcc tgac 24

<210> 10

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223>primers F 3

<400> 10

cccaagcttg ccgccaccat gacca 25

<210> 11

<211> 896

<212> DNA

<213> artificial sequence

<220>

<223>segment A

<400> 11

ggccggactg ctggcttctt ctagagccgc cacaaggcgg tactatctgg gcgctgtgga 60

actgtcatgg gattacatgc agtccgacct gggagagctg ccagtggacg ctcgatttcc 120

ccctcgtgtc cctaagagct tcccattcaa cacatctgtg gtctataaga aaacactgtt 180

cgtggagttt actgatcacc tgttcaacat cgccaagcct aggccaccct ggatgggact 240

gctgggacca actatccagg ctgaggtgta cgacaccgtg gtcattacac tgaaaaacat 300

ggcatcacac ccagtgtccc tgcatgccgt gggggtctct tactggaagg ctagtgaagg 360

tgcagagtat gacgatcaga catctcagcg ggaaaaagag gacgataagg tgtttcccgg 420

cggatcccat acttacgtgt ggcaggtcct gaaagagaat ggccccatgg cttctgatcc 480

tctgtgcctg acctactctt atctgagtca cgtggacctg gtcaaggatc tgaactccgg 540

gctgatcggt gcactgctgg tgtgcagaga agggagcctg gccaaggaga aaacccagac 600

actgcataag ttcattctgc tgttcgccgt gtttgacgag ggtaaatcat ggcactccga 660

gaccaagaac tccctgatgc aggacagaga tgcagctagc gcccgagctt ggccaaaaat 720

gcatacagtg aacggctacg tcaataggag tctgcccggc ctgatcggat gtcaccggaa 780

gtccgtgtat tggcatgtca tcgggatggg taccacaccc gaagtgcact ctattttcct 840

ggagggacat acctttctgg tccgaaacca ccgtcaggca agcctggaga tctgaa 896

<210> 12

<211> 69

<212> DNA

<213> artificial sequence

<220>

<223>segment B

<400> 12

aagcttgccg ccaccatgac cagactgacc gtgctggctc tgctggccgg actgctggct 60

tcttctaga 69

<210> 13

<211> 943

<212> DNA

<213> artificial sequence

<220>

<223>segment C

<400> 13

cccaagcttg ccgccaccat gaccagactg accgtgctgg ctctgctggc cggactgctg 60

gcttcttcta gagccgccac aaggcggtac tatctgggcg ctgtggaact gtcatgggat 120

tacatgcagt ccgacctggg agagctgcca gtggacgctc gatttccccc tcgtgtccct 180

aagagcttcc cattcaacac atctgtggtc tataagaaaa cactgttcgt ggagtttact 240

gatcacctgt tcaacatcgc caagcctagg ccaccctgga tgggactgct gggaccaact 300

atccaggctg aggtgtacga caccgtggtc attacactga aaaacatggc atcacaccca 360

gtgtccctgc atgccgtggg ggtctcttac tggaaggcta gtgaaggtgc agagtatgac 420

gatcagacat ctcagcggga aaaagaggac gataaggtgt ttcccggcgg atcccatact 480

tacgtgtggc aggtcctgaa agagaatggc cccatggctt ctgatcctct gtgcctgacc 540

tactcttatc tgagtcacgt ggacctggtc aaggatctga actccgggct gatcggtgca 600

ctgctggtgt gcagagaagg gagcctggcc aaggagaaaa cccagacact gcataagttc 660

attctgctgt tcgccgtgtt tgacgagggt aaatcatggc actccgagac caagaactcc 720

ctgatgcagg acagagatgc agctagcgcc cgagcttggc caaaaatgca tacagtgaac 780

ggctacgtca ataggagtct gcccggcctg atcggatgtc accggaagtc cgtgtattgg 840

catgtcatcg ggatgggtac cacacccgaa gtgcactcta ttttcctgga gggacatacc 900

tttctggtcc gaaaccaccg tcaggcaagc ctggagatct gaa 943

<210> 14

<211> 937

<212> DNA

<213> artificial sequence

<220>

<223>segment D

<400> 14

aagcttgccg ccaccatgac cagactgacc gtgctggctc tgctggccgg actgctggct 60

tcttctagag ccgccacaag gcggtactat ctgggcgctg tggaactgtc atgggattac 120

atgcagtccg acctgggaga gctgccagtg gacgctcgat ttccccctcg tgtccctaag 180

agcttcccat tcaacacatc tgtggtctat aagaaaacac tgttcgtgga gtttactgat 240

cacctgttca acatcgccaa gcctaggcca ccctggatgg gactgctggg accaactatc 300

caggctgagg tgtacgacac cgtggtcatt acactgaaaa acatggcatc acacccagtg 360

tccctgcatg ccgtgggggt ctcttactgg aaggctagtg aaggtgcaga gtatgacgat 420

cagacatctc agcgggaaaa agaggacgat aaggtgtttc ccggcggatc ccatacttac 480

gtgtggcagg tcctgaaaga gaatggcccc atggcttctg atcctctgtg cctgacctac 540

tcttatctga gtcacgtgga cctggtcaag gatctgaact ccgggctgat cggtgcactg 600

ctggtgtgca gagaagggag cctggccaag gagaaaaccc agacactgca taagttcatt 660

ctgctgttcg ccgtgtttga cgagggtaaa tcatggcact ccgagaccaa gaactccctg 720

atgcaggaca gagatgcagc tagcgcccga gcttggccaa aaatgcatac agtgaacggc 780

tacgtcaata ggagtctgcc cggcctgatc ggatgtcacc ggaagtccgt gtattggcat 840

gtcatcggga tgggtaccac acccgaagtg cactctattt tcctggaggg acataccttt 900

ctggtccgaa accaccgtca ggcaagcctg gagatct 937

<210> 15

<211> 6117

<212> DNA

<213> artificial sequence

<220>

<223>segment E

<400> 15

gatctctcct attaccttcc tgacagccca gactctgctg atggatctgg ggcagttcct 60

gctgttttgc cacatctcca gccaccagca tgatggtatg gaggcatacg tgaaagtgga 120

ctcctgtccc gaggaacctc agctgagaat gaagaacaat gaggaagccg aagactatga 180

cgatgacctg actgactccg agatggatgt ggtccgcttc gatgacgata actccccctc 240

cttcatccag attcgaagcg tggccaagaa acaccccaag acctgggtcc attacatcgc 300

agccgaggaa gaggactggg attatgcacc actggtgctg gcaccagacg acaggtccta 360

caaatctcag tatctgaaca atgggcctca gaggatcggt cggaagtaca agaaagtgcg 420

attcatggcc tataccgatg aaacatttaa gactcgtgaa gctatccagc acgagtccgg 480

gattctgggt ccactgctgt acggcgaagt gggagacact ctgctgatca tcttcaagaa 540

ccaggctagc aggccttaca atatctatcc acatggcatt accgatgtgc ggcctctgta 600

ctctagacgc ctgccaaagg gcgtcaaaca cctgaaggac ttcccaatcc tgcccggaga 660

aatcttcaag tataaatgga ctgtcaccgt cgaggatgga cccactaagt ctgaccctag 720

gtgcctgacc cggtactatt ctagtttcgt gaatatggaa agggatctgg cctccgggct 780

gatcggtcca ctgctgattt gttacaaaga gtctgtggat cagcgaggca accagatcat 840

gagtgacaag cgtaatgtga ttctgttctc cgtctttgac gaaaacagaa gctggtatct 900

gaccgagaac atccagaggt tcctgcctaa tccagcaggc gtgcagctgg aagatcccga 960

gtttcaggcc tcaaacatca tgcattccat taatggatac gtgttcgaca gtctgcagct 1020

gtcagtgtgc ctgcacgagg tcgcctactg gtatatcctg tccattgggg ctcagacaga 1080

tttcctgtcc gtgttcttta gcggttacac tttcaagcat aagatggtct acgaggacac 1140

actgactctg ttcccttttt ctggcgaaac cgtgtttatg agtatggaga atccaggcct 1200

gtggatcctg ggatgccaca acagcgattt ccgaaatcgt ggcatgactg ctctgctgaa 1260

agtgtcatcc tgtgacaaga acaccggaga ctactatgaa gatagctacg aggacatctc 1320

tgcttatctg ctgagtaaga acaatgcaat tgaacccagg tcttttagtc agaactctcg 1380

gcatcctagt cagaatcctc cagtgctgaa gagacaccag cgcgagatca ctagaactac 1440

cctgcagagt gatcaggaag agatcgacta cgacgatacc atttccgtgg aaatgaagaa 1500

agaggacttc gatatctatg acgaagatga gaaccagagt cctagatcat tccagaagaa 1560

aacccgccat tactttattg ctgcagtgga gcgcctgtgg gattatggga tgagctctag 1620

tccccacgtg ctgaggaatc gggcccagtc aggttccgtc cctcagttca agaaagtggt 1680

cttccaggag tttacagacg gcagctttac tcagcctctg tacagaggag aactgaacga 1740

gcacctgggc ctgctgggac catatatccg cgccgaagtg gaggataaca ttatggtcac 1800

cttccgaaat caggcttcac gtccctactc cttttattca tccctgatct cttacgaaga 1860

ggaccagcga cagggcgctg aaccccgtaa aaacttcgtg aagcctaatg agaccaaaac 1920

atacttttgg aaggtgcagc accatatggc ccctacaaaa gacgagttcg attgcaaggc 1980

atgggcctat ttttcagacg tggatctgga gaaggacgtg cactctgggc tgatcggtcc 2040

tctgctggtg tgccatacta acaccctgaa tccagctcac ggcaggcagg tgacagtcca 2100

ggagttcgca ctgttcttta ccatctttga tgagacaaag tcctggtact tcactgaaaa 2160

catggagcga aattgccgtg ctccatgtaa tattcagatg gaagacccca ccttcaagga 2220

gaactaccgg tttcatgcaa tcaatggcta tattatggat acactgccag gactggtgat 2280

ggcccaggac cagagaatcc gctggtacct gctgtccatg ggcagcaacg agaatatcca 2340

cagcattcat ttctctggac acgtgtttac agtccgcaag aaagaagagt ataaaatggc 2400

cctgtacaac ctgtatccag gggtgttcga aactgtcgag atgctgccca gcaaggctgg 2460

tatctggcga gtggaatgcc tgattgggga gcacctgcat gcaggcatgt caaccctgtt 2520

tctggtgtac tccaataagt gtcagacacc actggggatg gccagcggtc atatccgaga 2580

tttccagatt accgcttctg gccagtacgg acagtgggcc cccaagctgg ctaggctgca 2640

ctatagcggc tctatcaacg cctggagtac caaagagccc ttttcatgga ttaaggtgga 2700

cctgctggct cctatgatca ttcatgggat caaaacacag ggtgcacggc agaagttcag 2760

ctctctgtac atcagccagt tcatcatcat gtactctctg gatggaaaga aatggcagac 2820

atacagaggc aatagcacag gaactctgat ggtgttcttt ggcaacgtgg acagttcagg 2880

aatcaagcac aacattttca atccccctat cattgctagg tacatccggc tgcaccctac 2940

ccattattct attagaagta cactgcgcat ggaactgatg gggtgcgatc tgaacagttg 3000

ttcaatgcca ctgggtatgg agagtaaggc aatctcagac gcccagatta ccgcttccag 3060

ctacttcact aatatgtttg ctacctggtc ccctagcaaa gcaagactgc atctgcaggg 3120

caggagcaac gcatggcggc cacaggtgaa caatcccaag gagtggctgc aggtcgattt 3180

tcagaaaact atgaaggtga ccggcgtcac aactcaggga gtgaaatccc tgctgactag 3240

catgtatgtc aaggagttcc tgatctctag ttcacaggac ggacaccagt ggaccctgtt 3300

ctttcagaac gggaaggtga aagtcttcca gggtaatcag gatagtttta cacccgtggt 3360

caactcactg gacccacccc tgctgactag atacctgcgc atccaccctc agtcttgggt 3420

gcatcagatt gctctgagga tggaagtcct gggctgtgag gcacaggacc tgtattgata 3480

agaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 3540

ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 3600

ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt 3660

ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct 3720

gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct 3780

gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 3840

gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga 3900

tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca 3960

cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 4020

tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 4080

gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 4140

ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 4200

cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 4260

ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 4320

cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 4380

tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 4440

tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 4500

tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 4560

ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 4620

tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 4680

cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 4740

gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 4800

ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 4860

acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 4920

cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 4980

atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 5040

tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 5100

tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 5160

aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 5220

aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt 5280

taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 5340

taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 5400

agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 5460

tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 5520

cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 5580

agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 5640

gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 5700

aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 5760

tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 5820

ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 5880

aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 5940

ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 6000

agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 6060

gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgcca 6117

<210> 16

<211> 4413

<212> DNA

<213> artificial sequence

<220>

<223>segment F

<400> 16

agcttgccgc caccatgacc agactgaccg tgctggctct gctggccgga ctgctggctt 60

cttctagagc cgccacaagg cggtactatc tgggcgctgt ggaactgtca tgggattaca 120

tgcagtccga cctgggagag ctgccagtgg acgctcgatt tccccctcgt gtccctaaga 180

gcttcccatt caacacatct gtggtctata agaaaacact gttcgtggag tttactgatc 240

acctgttcaa catcgccaag cctaggccac cctggatggg actgctggga ccaactatcc 300

aggctgaggt gtacgacacc gtggtcatta cactgaaaaa catggcatca cacccagtgt 360

ccctgcatgc cgtgggggtc tcttactgga aggctagtga aggtgcagag tatgacgatc 420

agacatctca gcgggaaaaa gaggacgata aggtgtttcc cggcggatcc catacttacg 480

tgtggcaggt cctgaaagag aatggcccca tggcttctga tcctctgtgc ctgacctact 540

cttatctgag tcacgtggac ctggtcaagg atctgaactc cgggctgatc ggtgcactgc 600

tggtgtgcag agaagggagc ctggccaagg agaaaaccca gacactgcat aagttcattc 660

tgctgttcgc cgtgtttgac gagggtaaat catggcactc cgagaccaag aactccctga 720

tgcaggacag agatgcagct agcgcccgag cttggccaaa aatgcataca gtgaacggct 780

acgtcaatag gagtctgccc ggcctgatcg gatgtcaccg gaagtccgtg tattggcatg 840

tcatcgggat gggtaccaca cccgaagtgc actctatttt cctggaggga catacctttc 900

tggtccgaaa ccaccgtcag gcaagcctgg agatctctcc tattaccttc ctgacagccc 960

agactctgct gatggatctg gggcagttcc tgctgttttg ccacatctcc agccaccagc 1020

atgatggtat ggaggcatac gtgaaagtgg actcctgtcc cgaggaacct cagctgagaa 1080

tgaagaacaa tgaggaagcc gaagactatg acgatgacct gactgactcc gagatggatg 1140

tggtccgctt cgatgacgat aactccccct ccttcatcca gattcgaagc gtggccaaga 1200

aacaccccaa gacctgggtc cattacatcg cagccgagga agaggactgg gattatgcac 1260

cactggtgct ggcaccagac gacaggtcct acaaatctca gtatctgaac aatgggcctc 1320

agaggatcgg tcggaagtac aagaaagtgc gattcatggc ctataccgat gaaacattta 1380

agactcgtga agctatccag cacgagtccg ggattctggg tccactgctg tacggcgaag 1440

tgggagacac tctgctgatc atcttcaaga accaggctag caggccttac aatatctatc 1500

cacatggcat taccgatgtg cggcctctgt actctagacg cctgccaaag ggcgtcaaac 1560

acctgaagga cttcccaatc ctgcccggag aaatcttcaa gtataaatgg actgtcaccg 1620

tcgaggatgg acccactaag tctgacccta ggtgcctgac ccggtactat tctagtttcg 1680

tgaatatgga aagggatctg gcctccgggc tgatcggtcc actgctgatt tgttacaaag 1740

agtctgtgga tcagcgaggc aaccagatca tgagtgacaa gcgtaatgtg attctgttct 1800

ccgtctttga cgaaaacaga agctggtatc tgaccgagaa catccagagg ttcctgccta 1860

atccagcagg cgtgcagctg gaagatcccg agtttcaggc ctcaaacatc atgcattcca 1920

ttaatggata cgtgttcgac agtctgcagc tgtcagtgtg cctgcacgag gtcgcctact 1980

ggtatatcct gtccattggg gctcagacag atttcctgtc cgtgttcttt agcggttaca 2040

ctttcaagca taagatggtc tacgaggaca cactgactct gttccctttt tctggcgaaa 2100

ccgtgtttat gagtatggag aatccaggcc tgtggatcct gggatgccac aacagcgatt 2160

tccgaaatcg tggcatgact gctctgctga aagtgtcatc ctgtgacaag aacaccggag 2220

actactatga agatagctac gaggacatct ctgcttatct gctgagtaag aacaatgcaa 2280

ttgaacccag gtcttttagt cagaactctc ggcatcctag tcagaatcct ccagtgctga 2340

agagacacca gcgcgagatc actagaacta ccctgcagag tgatcaggaa gagatcgact 2400

acgacgatac catttccgtg gaaatgaaga aagaggactt cgatatctat gacgaagatg 2460

agaaccagag tcctagatca ttccagaaga aaacccgcca ttactttatt gctgcagtgg 2520

agcgcctgtg ggattatggg atgagctcta gtccccacgt gctgaggaat cgggcccagt 2580

caggttccgt ccctcagttc aagaaagtgg tcttccagga gtttacagac ggcagcttta 2640

ctcagcctct gtacagagga gaactgaacg agcacctggg cctgctggga ccatatatcc 2700

gcgccgaagt ggaggataac attatggtca ccttccgaaa tcaggcttca cgtccctact 2760

ccttttattc atccctgatc tcttacgaag aggaccagcg acagggcgct gaaccccgta 2820

aaaacttcgt gaagcctaat gagaccaaaa catacttttg gaaggtgcag caccatatgg 2880

cccctacaaa agacgagttc gattgcaagg catgggccta tttttcagac gtggatctgg 2940

agaaggacgt gcactctggg ctgatcggtc ctctgctggt gtgccatact aacaccctga 3000

atccagctca cggcaggcag gtgacagtcc aggagttcgc actgttcttt accatctttg 3060

atgagacaaa gtcctggtac ttcactgaaa acatggagcg aaattgccgt gctccatgta 3120

atattcagat ggaagacccc accttcaagg agaactaccg gtttcatgca atcaatggct 3180

atattatgga tacactgcca ggactggtga tggcccagga ccagagaatc cgctggtacc 3240

tgctgtccat gggcagcaac gagaatatcc acagcattca tttctctgga cacgtgttta 3300

cagtccgcaa gaaagaagag tataaaatgg ccctgtacaa cctgtatcca ggggtgttcg 3360

aaactgtcga gatgctgccc agcaaggctg gtatctggcg agtggaatgc ctgattgggg 3420

agcacctgca tgcaggcatg tcaaccctgt ttctggtgta ctccaataag tgtcagacac 3480

cactggggat ggccagcggt catatccgag atttccagat taccgcttct ggccagtacg 3540

gacagtgggc ccccaagctg gctaggctgc actatagcgg ctctatcaac gcctggagta 3600

ccaaagagcc cttttcatgg attaaggtgg acctgctggc tcctatgatc attcatggga 3660

tcaaaacaca gggtgcacgg cagaagttca gctctctgta catcagccag ttcatcatca 3720

tgtactctct ggatggaaag aaatggcaga catacagagg caatagcaca ggaactctga 3780

tggtgttctt tggcaacgtg gacagttcag gaatcaagca caacattttc aatcccccta 3840

tcattgctag gtacatccgg ctgcacccta cccattattc tattagaagt acactgcgca 3900

tggaactgat ggggtgcgat ctgaacagtt gttcaatgcc actgggtatg gagagtaagg 3960

caatctcaga cgcccagatt accgcttcca gctacttcac taatatgttt gctacctggt 4020

cccctagcaa agcaagactg catctgcagg gcaggagcaa cgcatggcgg ccacaggtga 4080

acaatcccaa ggagtggctg caggtcgatt ttcagaaaac tatgaaggtg accggcgtca 4140

caactcaggg agtgaaatcc ctgctgacta gcatgtatgt caaggagttc ctgatctcta 4200

gttcacagga cggacaccag tggaccctgt tctttcagaa cgggaaggtg aaagtcttcc 4260

agggtaatca ggatagtttt acacccgtgg tcaactcact ggacccaccc ctgctgacta 4320

gatacctgcg catccaccct cagtcttggg tgcatcagat tgctctgagg atggaagtcc 4380

tgggctgtga ggcacaggac ctgtattgat aag 4413

<210> 17

<211> 6893

<212> DNA

<213> artificial sequence

<220>

<223>segment G

<400> 17

aattcattga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac 60

ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg 120

tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 180

gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 240

gtctggcggc cgcgacctgc aggcgcagaa ctggtaggta tggaagatcc ctcgagatcc 300

attgtgctgg cggtaggcga gcagcgcctg cctgaagctg cgggcattcc cgatcagaaa 360

tgagcgccag tcgtcgtcgg ctctcggcac cgaatgcgta tgattctccg ccagcatggc 420

ttcggccagt gcgtcgagca gcgcccgctt gttcctgaag tgccagtaaa gcgccggctg 480

ctgaaccccc aaccgttccg ccagtttgcg tgtcgtcaga ccgtctacgc cgacctcgtt 540

caacaggtcc agggcggcac ggatcactgt attcggctgc aactttgtca tgcttgacac 600

tttatcactg ataaacataa tatgtccacc aacttatcag tgataaagaa tccgcgccag 660

cacaatggat ctcgaggtcg agggatctct agaggatcct ctacgccgga cgcatcgtgg 720

ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg 780

gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg 840

caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg 900

cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata 960

agggagagcg tcgacctcgg gccgcgttgc tggcgttttt ccataggctc cgcccccctg 1020

acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 1080

gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 1140

ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 1200

gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 1260

cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 1320

taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 1380

atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 1440

cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 1500

cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 1560

ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 1620

ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 1680

tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 1740

aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 1800

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 1860

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 1920

atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 1980

tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 2040

ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 2100

ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 2160

tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 2220

ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 2280

ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 2340

tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 2400

gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 2460

taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 2520

cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 2580

agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 2640

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 2700

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 2760

ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tgatggctct 2820

ttgcggcacc catcgttcgt aatgttccgt ggcaccgagg acaaccctca agagaaaatg 2880

taatcacact ggctcacctt cgggtgggcc tttctgcgtt tataaggaga cactttatgt 2940

ttaagaaggt tggtaaattc cttgcggctt tggcagccaa gctagatccg gctgtggaat 3000

gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 3060

atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga 3120

agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc 3180

atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 3240

tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 3300

ggcttttttg gaggcctagg cttttgcaaa aagctagctt ggggccaccg ctcagagcac 3360

cttccaccat ggccacctca gcaagttccc acttgaacaa aaacatcaag caaatgtact 3420

tgtgcctgcc ccagggtgag aaagtccaag ccatgtatat ctgggttgat ggtactggag 3480

aaggactgcg ctgcaaaacc cgcaccctgg actgtgagcc caagtgtgta gaagagttac 3540

ctgagtggaa ttttgatggc tctagtacct ttcagtctga gggctccaac agtgacatgt 3600

atctcagccc tgttgccatg tttcgggacc ccttccgcag agatcccaac aagctggtgt 3660

tctgtgaagt tttcaagtac aaccggaagc ctgcagagac caatttaagg cactcgtgta 3720

aacggataat ggacatggtg agcaaccagc acccctggtt tggaatggaa caggagtata 3780

ctctgatggg aacagatggg cacccttttg gttggccttc caatggcttt cctgggcccc 3840

aaggtccgta ttactgtggt gtgggcgcag acaaagccta tggcagggat atcgtggagg 3900

ctcactaccg cgcctgcttg tatgctgggg tcaagattac aggaacaaat gctgaggtca 3960

tgcctgccca gtgggagttc caaataggac cctgtgaagg aatccgcatg ggagatcatc 4020

tctgggtggc ccgtttcatc ttgcatcgag tatgtgaaga ctttggggta atagcaacct 4080

ttgaccccaa gcccattcct gggaactgga atggtgcagg ctgccatacc aactttagca 4140

ccaaggccat gcgggaggag aatggtctga agcacatcga ggaggccatc gagaaactaa 4200

gcaagcggca ccggtaccac attcgagcct acgatcccaa ggggggcctg gacaatgccc 4260

gtcgtctgac tgggttccac gaaacgtcca acatcaacga cttttctgct ggtgtcgcca 4320

atcgcagtgc cagcatccgc attccccgga ctgtcggcca ggagaagaaa ggttactttg 4380

aagaccgccg cccctctgcc aattgtgacc cctttgcagt gacagaagcc atcgtccgca 4440

catgccttct caatgagact ggcgacgagc ccttccaata caaaaactaa ttagactttg 4500

agtgatcttg agcctttcct agttcatccc accccgcccc agagagatct ttgtgaagga 4560

accttacttc tgtggtgtga cataattgga caaactacct acagagattt aaagctctaa 4620

ggtaaatata aaatttttaa gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt 4680

attttagatt ccaacctatg gaactgatga atgggagcag tggtggaatg cctttaatga 4740

ggaaaacctg ttttgctcag aagaaatgcc atctagtgat gatgaggcta ctgctgactc 4800

tcaacattct actcctccaa aaaagaagag aaaggtagaa gaccccaagg actttccttc 4860

agaattgcta agttttttga gtcatgctgt gtttagtaat agaactcttg cttgctttgc 4920

tatttacacc acaaaggaaa aagctgcact gctatacaag aaaattatgg aaaaatattc 4980

tgtaaccttt ataagtaggc ataacagtta taatcataac atactgtttt ttcttactcc 5040

acacaggcat agagtgtctg ctattaataa ctatgctcaa aaattgtgta cctttagctt 5100

tttaatttgt aaaggggtta ataaggaata tttgatgtat agtgccttga ctagagatca 5160

taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc 5220

ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt 5280

ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 5340

tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatctcta 5400

gcttcgtgtc aaggacggtg aggcgcgcct actgagtcat tagggacttt ccaatgggtt 5460

ttgcccagta cataaggtca ataggggtga atcaacagga aagtcccatt ggagccaagt 5520

acactgagtc aatagggact ttccattggg ttttgcccag tacaaaaggt caataggggg 5580

tgagtcaatg ggtttttccc attattggca cgtacataag gtcaataggg gtgagtcatt 5640

gggtttttcc agccaattta attaaaacgc catgtacttt cccaccattg acgtcaatgg 5700

gctattgaaa ctaatgcaac gtgaccttta aacggtactt tcccatagct gattaatggg 5760

aaagtaccgt tctcgagcca atacacgtca atgggaagtg aaagggcagc caaaacgtaa 5820

caccgccccg gttttcccct ggaaattcca tattggcacg cattctattg gctgagctgc 5880

gttctacgtg ggtataagag gcgcgaccag cgtcggtacc gtcgcagtct tcggtctgac 5940

caccgtagaa cgcagcctca ggacctccat agaagacacc gggaccgatc cagcctccgc 6000

ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt aagtaccgcc 6060

tatagagtct ataggcccac ccccttggct tcttatgcat gctatactgt ttttggcttg 6120

gggtctatac acccccgctt cctcatgtta taggtgatgg tatagcttag cctataggtg 6180

tgggttattg accattattg accactcccc tattggtgac gatactttcc attactaatc 6240

cataacatgg ctctttgcca caactctctt tattggctat atgccaatac actgtccttc 6300

agagactgac acggactctg tatttttaca ggatggggtc tcatttatta tttacaaatt 6360

cacatataca acaccaccgt ccccagtgcc cgcagttttt attaaacata acgtgggatc 6420

tccacgcgaa tctcgggtac gtgttccgga catgggctct tctccggtag cggcggagct 6480

tctacatccg agccctgctc ccatgcctcc agcgactcat ggtcgctcgg cagctccttg 6540

ctcctaacag tggaggccag acttaggcac agcacgatgc ccaccaccac cagtgtgccg 6600

cacaaggccg tggcggtagg gtatgtgtct gaaaatgagc tcggggagcg ggcttgcacc 6660

gctgacgcat ttggaagact taaggcagcg gcagaagaag atgcaggcag ctgagttgtt 6720

gtgttctgat aagagtcaga ggtaactccc gttgcggtgc tgttaacggt ggagggcagt 6780

gtagtctgag cagtactcgt tgctgccgcg cgcgccacca gacataatag ctgacagact 6840

aacagactgt tcctttccat gggtcttttc tgcagtcacc gtccttgaca cga 6893

Claims (10)

1. a kind of method for improving recombinant human blood coagulation factor VII I high efficient expression, comprising the following steps:

(1) building of recombination, the recombination include signal peptide gene and rhFVIII gene；

(2) above-mentioned recombination segment is inserted into plasmid, obtains recombinant plasmid；

(3) above-mentioned recombinant plasmid is subjected to PvuI linearisation, electricity is gone in mammalian cell, identified and determination of activity, sieve Select positive cell strain；

(4) by it is above-mentioned screen positive cell strain is seeded in cell culture medium, cultivate 4-5 days, added into cell culture medium External source fetal calf serum collects culture solution and carries out determination of activity；It is characterized in that, the additional amount of the fetal calf serum is 2-6%.

2. the method according to claim 1, wherein the nucleotides sequence of rhFVIII is classified as in the step (1) SEQ ID NO:1.

3. the method according to claim 1, wherein signal peptide is reddish black killing after optimization in the step (1) Former albumen (Azu) before element, nucleotides sequence are classified as SEQ ID NO:4, and obtained recombination is Azu-rhFVIII, nucleotides sequence It is classified as SEQ ID NO:6.

4. according to the method described in claim 3, it is characterized in that, the amino acid sequence of the recombination Azu-rhFVIII For SEQ ID NO:7.

5. being obtained the method according to claim 1, wherein the plasmid in the step (2) is pXC17.4 Recombinant expression plasmid is pXC17.4-Azu-rhFVIII.

6. the method according to claim 1, wherein the mammalian cell in the step (3) is Chinese storehouse Mouse egg cell CHOK1SV-KO.

7. the method according to claim 1, wherein cell culture medium is CD CHO AGT in the step (4) Culture medium.

8. the method according to claim 1, wherein the additional amount of fetal calf serum is 3- in the step (4) 5%.

9. the method according to claim 1, wherein the additional amount of fetal calf serum is 3% in the step (4).

10. the method according to claim 1, wherein in the step (3) positive cell strain screening technique Are as follows: electricity is turned into cell inoculation into serum free medium, after 35-37 DEG C is cultivated 1 day, supernatant is abandoned in centrifugation, is added toward cell precipitation Enter preheated culture medium (50 μm of ol/L MSX CD CHO AGT culture mediums) resuspension, re-suspension liquid is transferred to conical flask and continues to train It supports, cultivates to after 10 days, sampling screens to obtain positive cell strain CHOK1SV-KO/pXC17.4-Azu- by PCR and activity identification rhFVIII。